This project is concerned with the mechanism of action of thymidylate synthetase (deoxyuridylate plus 5,10- methylenetetrahydrofolate yields thymidylate plus dihydrofolate) from amethopterin-resistant Lactobacillus casei. The problems to be investigated are (a) activation by thiols; (b) sulfhydryl group interactions; (c) probe of possible esterase activity of thymidylate synthetase and models thereof; (d) tryptophan titrations; (e) kinetic binding studies; (f) hydrogen exchange at C-5 of deoxyuridylate and models thereof; (g) formation and interaction of substituted methylene tetrahydrofolates with thymidylate synthetase; (h) chemical modification and identification of active site functional groups; (i) characterization of subunit structure and relevance to catalytic activity; and (j) cellular studies to determine the effect of amethopterin resistance on the nature and level of thymidylate synthetase and selected enzymes.